Our long term goal is to develop cellular based immunotherapy for the treatment of malignancy. One approach to enhancing the effects of cellular immunotherapy is via gene therapy. We are currently focused on enhancing the antitumor effects of adoptive T cell therapy using lentiviral vector-mediated genetic modification of cells to express a universal T cell receptor (TCR), or chimeric immune receptor (CIR). CIRs are comprised of an extracellular antibody fragment specific to a tumor antigen linked to an intracellular T cell activation domain capable of providing the cell with MHC unrestricted antigen-specific activation. Current treatments for B cell malignancies include chemotherapy, radiation therapy, bone marrow transplantation, and peripheral blood stem cell transplantation. Despite these treatment modalities, most patients will remain incurable. Cell based immunotherapy offers the promise of an alternative treatment option for patients who have tried and failed presently available treatment options. We have developed a CIR targeting the pan-B cell marker CD19. In comparison to the CD3-( alone signaling chain, we will test the multipartite signaling domains 41BB-( and CD28:41BB:( in our aCD19 CIR. Our specific goal in the proposed studies is to establish in preclinical (phase I) and an exploratory clinical study (phase II) the optimal signaling chain for CIR immunotherapy for B cell malignancy, using competitive repopulation experiments to identify the best candidate for commercialization of the therapy. Optimizing safety is a critical step towards a commercially feasible gene-based immunotherapy for cancer. One way to improve the safety of CIR T cells is to include a suicide gene switch that in the case of CIR-mediated toxicity will facilitate specific deletion of gene-modified cells by chemotherapy that is a low enough dose to leave an in tact immune system. A new class of suicide genes isolated from drosophila melanogaster, called dNK (for deoxyribonucleoside kinase) have been developed which offer altered substrate activity that is not influenced by the antivirals commonly used in cancer patients. We hypothesize that expression of the suicide gene will not alter the ability of CIR engineered cells to control tumors and that it will permit selective killing of the engineered cell upon introduction of substrate chemotherapy. In Aim 1, we will build and test the function of single and multipartite signaling chain CIR constructs with the dNK suicide gene in vitro; in Aim 2 we will evaluate the function of these CIRs and dNK suicide gene in a competitive repopulation in vivo in the NOG mouse model. [unreadable] [unreadable] [unreadable] [unreadable]